雄激素受体在肝癌发生过程中的表达及意义

为了进一步探讨雄激素受体（ＡｎｄｒｏｇｅｎＲｅｃｅｐｔｏｒ,ＡＲ）作为肝癌标志物的意义,本文采用免疫组织化学ＡＢＣ法,对二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌发生过程中肝细胞雄激素受体（ＡＲ）的表达进行了系统观察。结果显示：正常大鼠的ＡＲ阳性肝细胞极少,ＤＥＮ诱癌第４周可见少量肝细胞呈ＡＲ阳性表达,细胞散在分布,胞质和／或胞核内可见棕褐色阳性反应颗粒。随着肝癌发展进程,ＡＲ阳性肝细胞数逐渐增多,呈簇状或片状分布。至诱癌第１８周,肝癌结节内肝癌细胞大多呈ＡＲ阳性表达。本实验结果表明,ＡＲ与肝癌的发生和发展具有密切关系     

大鼠肝癌发生过程中p53的突变和甲胎蛋白的表达

采用免疫组织化学ＡＢＣ和ＰＡＰ法,对二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌发生过程中突变型ｐ５３蛋白（ｍｐ５３）和甲胎蛋白（ＡＦＰ）在肝细胞中的表达进行了系统观察。结果显示：（１）ＤＥＮ诱发大鼠肝癌发生率为１００％;（２）正常大鼠及诱癌第４周大鼠的肝细胞均不表达ｍｐ５３,至诱癌第８周,可见少量肝细胞表达ｍｐ５３,诱癌晚期的癌结节内大部分肝癌细胞呈ｍｐ５３阳性表达,ｍｐ５３免疫反应阳性产物为胞核内棕褐色颗粒;（３）正常大鼠肝细胞不表达ＡＦＰ,诱癌早期（４～８周）的大鼠肝小叶内可见少量ＡＦＰ阳性肝细胞,多为小肝细胞,呈散在分布,此后ＡＦＰ阳性肝细胞逐渐增多,晚期的癌结节内大部分癌细胞呈ＡＦＰ阳性,ＡＦＰ免疫反应阳性产物为胞浆内棕褐色颗粒。结果提示,ｍｐ５３和ＡＦＰ可作为分析肝癌进展的病理学指标     

大鼠肝癌发生过程中转化生长因子-β1的表达及意义

为了进一步探讨转化生长因子-β1在肝癌发生中的作用和意义。本采用免疫组织化学ABC法,对二乙基亚硝胺（DEN）诱发大鼠肝癌发生过程中转化生长因子-β1（Transforming growth factor-β1,TGF-β1）表达情况进行了观察。结果显示;在正常大鼠肝脏,TGF-β1的表达只局限于血窦内皮细胞或枯否细胞,汇管区血管内皮细胞及胆管上皮细胞,肝细胞呈TGF-β1阴性表达,诱癌早期（4-8周）,大鼠肝小叶内除血窦内皮细胞或枯否细胞呈TGF-β1阳性表达外,可见少量散在分布的肝细胞呈TGF-β1阳性表达,阳性肝细胞胞质内可见棕褐色阳性反应颗粒,随着肝癌发展,TGF-β1阳性肝细胞逐渐增多,至诱癌晚期（18周）,癌结节内的大多数肝癌细胞呈TGF-β1阳性表达。本研究显示TGF-β1与肝癌的发生和发展密切相关。至诱癌晚期（18周）,癌结节内的大多数肝癌细胞呈TGF-β1阳性表达,本研究显示TGF-β1与肝癌的发生和发展密切相关。     

大鼠肝癌发生过程中NF-κB的表达

目的探讨核转录因子-κB(NF-κB)在大鼠肝癌发生发展中的作用和意义。方法应用免疫组织化学SP法,对二乙基亚硝胺(DEN)诱发的大鼠肝癌发生过程中NF-κB的动态表达进行了检测。结果 DEN诱发的肝癌为肝细胞癌,诱癌率为100%,大鼠肝癌癌变过程大致经过肝细胞损伤期、肝细胞增生-硬化期和肝细胞癌变期等三个阶段。在正常大鼠肝组织,偶见少量肝细胞呈阳性表达,随着肝癌发生发展,NF-κB阳性表达细胞逐渐增多,至诱癌晚期,可见大量NF-κB阳性表达细胞,均比正常肝组织表达高(P<0.05)。结论本研究表明肝细胞NF-κB的过度表达与肝癌的发生和发展密切有关。     

大鼠肝癌发生过程中增殖细胞核抗原（PCNA／Cyclin）的表达

应用免疫组织化学的ＡＢＣ法,对二乙基亚硝胺（ＤＥＮ）诱发大鼠发生过程中增殖细胞核抗原在肝组织中的表达进行了系统观察。结果显示：正常大鼠肝组织中仅见极少数ＰＣＮＡ阳性肝细胞,阳性率为０．０８％,随着诱癌进程发展,大鼠肝组织中ＰＣＮＡ阳性肝细胞逐渐增多,诱癌第４、８、１２周,大鼠肝组织中ＰＣＮＡ阳性肝细胞百分率分别为１．６％、３．８％、１６．２％,诱癌晚期癌结节内大部分肝癌细胞里ＰＣＮＡ阳性表达,阳性率为８０．６％。本研究结果表明原位检测ＰＣＮＡ表达比传统依据形态学分化程度来判断肿瘤发生可能性更为客观、可靠。     

MDM2在原发性肝细胞癌中的表达

目的检测MDM2蛋白及其mRNA在原发性肝细胞癌中的表达,探讨MDM2在原发性肝细胞癌发生发展中作用及意义.方法采用免疫组织化学、原位分子杂交和细胞图像分析技术,检测肝细胞癌组织及其对应癌旁肝组织(各20例),正常肝组织(5例)中MDM2蛋白及其mRNA 表达情况.结果在肝细胞癌组织、癌旁肝组织和正常肝组织中,MDM2蛋白免疫组织化学阳性反应颗粒的平均光密度分别为:0.404±0.105, 0.302±0.067, 0.087±0.034.肝细胞癌组织与癌旁肝组织、正常肝组织相比,差异均有显著性意义(P<0.05).MDM2 mRNA在肝细胞癌组织中呈阳性表达,而癌旁肝组织及正常肝组织均呈阴性.结论 MDM2过度表达在原发性肝细胞癌的发生发展过程中可能发挥了重要作用.     

在大鼠肝癌发生过程中GABA-BR1的表达变化及意义

为探讨γ-氨基丁酸B型受体1(γ-aminobutyric acid type B1 receptor,GABA-BR1)与肝癌发生的关系,利用二乙基亚硝胺(DEN)制备诱发型大鼠肝癌模型,模拟人类肝癌发生发展过程,采用免疫组织化学技术对GABA-BR1和细胞周期素E(Cyclin E)蛋白在大鼠肝脏中的表达进行定性和定量分析.结果显示,在正常肝组织中有少量散在分布的GABA-BR1表达阳性的细胞;在变性或坏死灶周围GABA-BR1表达阳性的肝细胞较多;增生结节中部分肝细胞呈过表达;在肝癌细胞中广泛表达.Cyclin E与GABA-BR1阳性表达的细胞分布特点相似.实验结果提示,在肝癌发生发展过程GABA-BR1过表达,可能对肝细胞的异常增生或癌变过程起到抑制作用.     

实验性肝癌糖原和癌基因N-ras表达的研究

通过应用原位杂交和组织化学技术,对二乙基亚硝胺诱发的大鼠肝细胞肝癌中糖原和癌基因Ｎ－ｒａｓ表达的研究,发现从诱癌早期到晚期,肝细胞内的糖原由储积而逐渐丧失。Ｎ－ｒａｓ在诱癌的第１～２周即出现阳性表达,随诱癌过程的延长,阳性表达的细胞数和范围逐渐增加,至诱癌晚期甚至在癌结节内均转为阴性。对肝组织连续切片中糖原和Ｎ－ｒａｓ表达的对比观察发现,糖原ＰＡＳ反应与Ｎ－ｒａｓ反应同步,糖原ＰＡＳ反应具有与Ｎ－ｒａｓ一致的异质性,其阳性与阴性病变分布与Ｎ－ｒａｓ表达重叠。提示Ｎ－ｒａｓ基因表达可能在肝癌的启动过程中发挥重要作用,并且可能涉及对糖原基因的调控。     

Sonic hedgehog在二乙基亚硝胺诱发大鼠肝癌发生过程中的表达及意义

观察肝脏组织中Sonic hedgehog(Shh)的表达情况,探讨其在肝癌发生发展过程中的作用.用二乙基亚硝胺(diethylnitrosamine,DEN)制备诱发型肝癌模型,利用光镜技术观察诱癌过程中肝组织的形态学改变,采用免疫组织化学二步法和RT-PCR技术检测Shh蛋白和mRNA的表达.根据形态学观察将诱癌过程分为正常对照组、肝损伤组、肝增生-硬化组和肝癌变组.Shh蛋白阳性表达的细胞主要分布在小叶间胆管上皮、肝细胞增生结节、癌周组织和癌结节中,在对照组、肝损伤期、肝增生-硬化期和肝癌变期的阳性表达率分别是6.67%、30.00%、52.94%和78.57%(χ2=17.49,P<0.05).Shh mRNA表达率随着肝癌的发生发展有逐渐增高的趋势(χ2=13.35,P<0.05),对Shh mRNA表达阳性的电泳条带进行图像分析结果显示Shh mRNA表达量随着肝癌的发生发展逐渐增高(F=110.26,P<0.05).Ptch mRNA表达率随着肝癌的发生发展有逐渐增高的趋势(χ2=19.83,P<0.05),对Ptch mRNA表达阳性的电泳条带进行图像分析结果显示Ptch mRNA表达量随着肝癌的发生发展逐渐增高(F=68.28,P<0.05).实验结果提示,Shh在肝癌发生发展过程中出现了异常活动,导致Shh信号通路激活并作用于肝的细胞,参与了诱导肝癌的发生发展.     

大鼠诱发型肝癌发生发展过程中SonicHedgehog、Ptc和Gli1表达的改变

目的 观察大鼠诱发肝癌过程中Sonic Hedgehog（Shh）信号通路相关基因的表达变化,探讨其在肝癌发生发展过程中的作用.方法 雄性Wistar大鼠48只,随机分成4组,利用二乙基亚硝胺（DEN）制备诱发性大鼠肝癌模型,应用原位核酸杂交技术检测Shh、Ptc、Gli1 mRNA在对照组、模型组（6w）、模型组（14w）、模型组（22w）大鼠肝脏癌变过程中的表达变化.结果 在模型组（6w）大鼠肝脏的肝小叶周边可见嗜酸性、气球样变性等肝细胞损伤的表现,模型组（14w）大鼠肝脏中可见肝假小叶和非典型增生结节,模型组（22w）大鼠肝脏中可见到高分化的肝细胞癌结节.Shh、Ptc、Gli1 mRNA阳性表达细胞主要分布在大鼠肝脏中的肝细胞损伤区、增生结节、癌结节、小叶间胆管上皮和癌旁组织中,Shh、Ptc、Gli1 mRNA在模型组的大鼠肝组织中表达的平均光密度值均高于对照组.结论 Shh信号通路在诱癌过程中异常激活,可能促进肝损伤后的正常修复、异常增殖及肝细胞癌变过程.     

化学诱发大鼠肝癌的亚细胞组分中两类丝氨酸蛋白激酶的动态变化

用二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌,隔周测定肝脏胞液、膜性和胞核中的蛋白激酶Ａ（ＰＫＡ）和蛋白激酶Ｃ（ＰＫＣ）的活力。发现胞液ＰＫＡ在诱癌过程中活力改变不大,胞液ＰＫＣ则逐步增高,在第１３周和２０周形成两个活力高峰。膜性ＰＫＡ和ＰＫＣ都呈双相变化,即在癌前期（１０－１４周）增加,癌形成期（１７－２０周）反而降至正常以下,胞核ＰＫＡ和ＰＫＣ也都在癌前期升至高峰,而癌形成期则低于癌前期,但仍高于正常（ＰＫＡ）或接近正常（ＰＫＣ）。因只有膜性ＰＫＣ在大鼠老化时降低,故这些变化不是鼠龄变化的结果,而是ＤＥＮ诱癌所引起,其变化机理可能与下降调节、细胞内转位或两型同工酶相反的升降变化有关。     

白藜芦醇通过糖代谢重编程抑制DEN诱导大鼠肝细胞癌前阶段的恶性增殖

白藜芦醇(resveratrol,RES)可抑制肝癌细胞的生长与增殖。但其在癌前阶段的作用尚不十分清楚。本文研究白藜芦醇对二乙基亚硝胺(diethylinitrosamine, DEN)诱导大鼠肝癌前阶段的作用及机制。SD大鼠分为正常对照组、RES处理组、DEN处理组和RES-DEN处理组。研究结果表明,DEN处理大鼠8周时,肝细胞的总增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)升高至2倍(P<0.05),核内PCNA蛋白表达水平升高至3倍(P<0.001),而RES-DEN处理组大鼠肝细胞总PCNA(P<0.05)和核内PCNA蛋白表达水平(P<0.001)降低。结果提示,RES可显著抑制肝细胞恶性增生。通过非靶向代谢物组学及代谢通路富集分析,结果表明,RES-DEN处理大鼠的肝细胞中,虽然磷酸戊糖途径向糖酵解途径的转变增强,但相较于DEN组大鼠,糖酵解水平并未出现显著提高,提示磷酸烯醇式丙酮酸-丙酮酸-乳酸这条代谢途径被抑制。进一步验证发现,这条代谢途径上的关键酶M2型丙酮酸激酶(M2-type pyruvate kinase,PKM2)和乳酸脱氢酶(lactate dehydrogenase,LDHA)蛋白质表达水平被抑制(P<0.05)。RES可通过调节糖代谢重编程,在肝癌的癌前阶段抑制DEN诱导的大鼠肝细胞的过度增殖,为RES预防肝癌提供了实验依据。     

1alpha,25-Dihydroxyvitamin D(3) inhibits rat liver ultrastructural changes and the development of gamma-glutamyltranspeptidase-positive foci in diethylnitrosamine-initiated and streptozotocin-induced diabetes-promoted hepatocarcinogenesis

In the present study, the chemopreventive effect of the active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (VD(3)), against chemically-induced and diabetes-promoted rat liver carcinogenesis was investigated. Hepatocarcinogenesis was initiated with a single intraperitoneal (i.p.) injection of diethylnitrosamine (DEN) (125 mg kg(-1) body weight) at week 4 followed by promotion with streptozotocin (STZ) (65 mg kg(-1) body weight with a single i.p. injection) at week 7. With this basic experimental regimen, the effect of VD(3) (0.3 microg (0.1 ml)(-1) propylene glycol per os twice a week) was investigated with effect from 4 weeks prior to the exposure of DEN. The results showed that VD(3) supplementation throughout the experimental period reduced the incidence, total number and multiplicity and altered the size of visible persistent nodules (PNs) in DEN- or DEN + STZ-treated rats as compared with their respective controls. In these two groups, it also caused a significant decrease in the number (p < 0.002 and 0.001 respectively) and focal area (p < 0.05) of gamma-glutamyltranspeptidase (GGT)-positive hepatic foci. Moreover, continuous supplementation of VD(3) exhibits a protective effect in maintaining the normal cellular architecture of the hepatocytes in DEN- or DEN + STZ-treated rats. Our results thus strongly suggest that VD(3) is very effective in the inhibition of DEN-initiated and STZ-induced diabetes-promoted rat liver carcinogenesis.     

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-alpha), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were collected to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-alpha immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN.     

二乙基亚硝胺诱导大鼠肝癌组织CLDN1基因表达及其甲基化

目的探讨二乙基亚硝胺（diethylnitrosamine,DEN）诱导大鼠肝癌发生中肝癌组织CLDN1基因表达及其启动子甲基化的规律。方法65只雄性Wistar大鼠随机选择40只作为模型组,其余作为正常组。模型组在1-12周饮用含DEN80mg／L的饮水以诱癌（每日8mg／kg）,各组在造模过程的第4周、8周、12周、16周随机5只取肝,第20周剩余大鼠取肝,应用RT-PCR方法检测肝组织CLDN1mRNA的表达,应用MSP法检测肝组织CLDN1启动子甲基化和非甲基化。结果模型大鼠病死率为10％（4／40）,正常组无死亡。至第20周,成瘤率达到100％。RT—PCR显示,与正常组比较,模型组在16周和20周CLDN1 mRNA表达下调（P〈0．05）,其他各周两组差异不显著。MSP结果表明,模型组肝组织CLDN1甲基化率达77．78％,而正常肝组织甲基化率为24％,两者比较差异有显著性（P〈0．01）。结论CLDN1启动子甲基化及CLDN1基因表达下调与大鼠肝癌病变相关,对其机制值得进一步深入研究。     

Potential chemoprevention of diethylnitrosamine-initiated and 2-acetylaminofluorene-promoted hepatocarcinogenesis by zerumbone from the rhizomes of the subtropical ginger (Zingiber zerumbet)

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P < 0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P < 0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P < 0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P < 0.05) lowered. By the TUNEL assay, there were significantly (P < 0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers.     

Combined supplementation of vanadium and 1alpha,25-dihydroxyvitamin D3 inhibit placental glutathione S-transferase positive foci in rat liver carcinogenesis

The combined effects of vanadium (V) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in inhibiting diethylnitrosamine (DEN)-induced and phenobarbital (PB) promoted hepatocarcinogenesis were examined in male Sprague-Dawley rats. All the rats were subjected to 70% partial hepatectomy (PH) at week 4 and 24h later were administered either solvent trioctanoin (Group B, D, F and H) or 10 mg DEN/kg (Group A, C, E and G) by gavage. Briefly after two weeks of DEN administration, PB were administered (0.05% in basal diet) to all the DEN-treated rats and continued till the completion of the experiment. Supplementary V at the dose of 0.5 ppm in drinking water ad libitum (Group C and D), 1,25(OH)2D3 at the dose of 3 microg/ml in propylene glycol per os twice a week (Group E and F) or both V and 1,25(OH)2D3 at the same above given doses (Group G and H) were started 4 weeks prior to DEN administration (week 0) and continued thereafter till week 15. The expression of the number and area of altered hepatocyte foci (AHF) positive for placental glutathione S-transferase (GST-P) was maximum in DEN-treated and PB promoted group (Group A). V (Group C) and 1,25(OH)2D3 (Group E) treatment significantly reduced the expression of GST-P-positive hepatocytes by 36.02% and 45.16% respectively but an additive protective action (61.46%) was found in Group G which received both V and 1,25(OH)2D3 for the entire period of the study. Moreover, histopathological examination and the incidence of hepatic hyperplastic nodules showed that combined action of V and 1,25(OH)2D3 can able to minimize the appearance of nodules as well and maintain the normal cellular architecture than V and 1,25(OH)2D3 when given alone. These results suggest that, when given together V and 1,25(OH)2D3 could be the chemopreventive agents for rat liver carcinogenesis.